Polymerase chain reaction (PCR) is a method of amplifying a nucleic acid wherein a target DNA fragment can be exponentially amplified by repeatedly carrying out dissociation of a DNA chain into a single strand, subsequent bonding, to the DNA strand, of a primer corresponding to a specific region in the strand, and DNA synthesis reaction with a DNA polymerase. In addition to the PCR method, there are nucleic acid amplification methods such as RT-PCR (reverse transcriptase-polymerase chain reaction), NASBA (nucleic acid sequence based amplification), LAMP (loop mediated isothermal amplification of DNA), TMA (transcription mediated amplification method) and 3SR (self-sustained sequence replication).
The amplification reaction of a nucleic acid in the nucleic acid amplification methods mentioned above is susceptible to the influence of a substance inhibiting the nucleic acid amplification reaction, such as a protein (hereinafter, referred to an inhibitor) contained in a biological sample, and the nucleic acid amplification reaction is inhibited by the inhibitor. Accordingly, the operation of extracting or purifying a nucleic acid component such as DNA or RNA from a biological sample is necessary prior to detection of a nucleic acid by the nucleic acid amplification method. However, the operation of extracting or purifying a nucleic acid component is troublesome and time-consuming. As methods capable of amplifying a target nucleic acid without conducting the operation of extracting or purifying a nucleic acid component, methods described in US Patent Application Laid-Open No. 2005/0089857 or International Laid-Open No. 00/08136 are known.
US Patent Application Laid-Open No. 2005/0089857 supra describes a method of amplifying a target nucleic acid, which comprises treating a biological sample with a solution containing a salt interacting with an inhibitor in order to reduce the influence of the inhibitor.
International Laid-Open No. 00/08136 supra describes a method of amplifying a nucleic acid from a microorganism present in a sample such as feces, which comprises washing the sample with an organic solvent to remove an inhibitor. Specifically, when the sample is feces, the feces are suspended in a suitable buffer solution, and the resulting suspension is centrifuged to remove large solids, and its supernatant is collected. The obtained supernatant is centrifuged, and the resulting supernatant is discarded, and the residual precipitates are washed by adding an organic solvent and centrifuging the precipitates, and microorganisms are recovered as the precipitates and used as a sample for nucleic acid amplification. International Laid-Open No. 00/08136 supra describes that hydrophilic organic solvents such as ethanol, methanol, 2-propanol, propanone (acetone), ethane nitrile (acetonitrile) and dimethyl sulfoxide (DMSO) or amphiphatic organic solvents such as butanol, 2-butanol and ethyl acetate can be used as the organic solvent. International Laid-Open No. 00/08136 also describes that the sample may be tissues collected surgically from the living body, a solid sample such as a tissue is desirably homogenized to facilitate washing, and a nucleic acid as the subject of amplification is not particularly limited to a gene of a pathogenic microorganism or a gene derived from the living body.
In the method in International Laid-Open No. 00/08136, however, a tissue derived from a living body is used as the sample, and when a nucleic acid derived from the tissue but not a nucleic acid in a microorganism present in the tissue is to be amplified, there arises a problem that when the homogenized tissue is washed with the above-mentioned organic solvent, the objective nucleic acid is removed together with an inhibitor, thus reducing the nucleic acid which can be recovered.